Matrix metalloproteinases: their functional role in lung cancer.

Lung malignancy is the foremost cause of cancer-related deaths globally and is frequently related to long-term tobacco smoking. Recent studies reveal that the expression of matrix metalloproteinases (MMPs) is extremely high in lung tumors compared with non-malignant lung tissue. MMPs are zinc-dependent proteases and are involved in the degradation of extracellular matrix (ECM). Several investigations have shown that MMPs manipulate the activity of non-ECM molecules, including cytokines, growth factors and receptors that control the tumor microenvironment. In this review, we have summarized and critically reviewed the published works on the role of MMPs in non-small-cell lung cancer. We have also explored the structure of MMPs, their various types and roles in lung cancer metastasis including invasion, migration and angiogenesis.

Molecular mechanisms underlying the divergent roles of SPARC in human carcinogenesis.

Communication between the cell and its surrounding environment, consisting of proteinaceous (non-living material) and extracellular matrix (ECM), is important for biophysiological and chemical signaling. This signaling results in a range of cellular activities, including cell division, adhesion, differentiation, invasion, migration and angiogenesis. The ECM non-structural secretory glycoprotein called secreted protein, acidic and rich in cysteine (SPARC), plays a significant role in altering cancer cell activity and the tumor's microenvironment (TME). However, the role of SPARC in cancer research has been the subject of controversy. This review mainly focuses on recent advances in understanding the contradictory nature of SPARC in relation to ECM assembly, cancer cell proliferation, adhesion, migration, apoptosis and tumor growth.

SPARC expression induces cell cycle arrest via STAT3 signaling pathway in medulloblastoma cells.

Dynamic cell interaction with ECM components has profound influence in cancer progression. SPARC is a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. We previously reported that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we demonstrate that expression of SPARC inhibits medulloblastoma cell proliferation. MTT assay indicated a dose-dependent reduction in tumor cell proliferation in adenoviral mediated expression of SPARC full length cDNA (Ad-DsRed-SP) in D425 and UW228 cells. Flow cytometric analysis showed that Ad-DsRed-SP-infected cells accumulate in the G2/M phase of cell cycle. Further, immunoblot and immunoprecipitation analyses revealed that SPARC induced G2/M cell cycle arrest was mediated through inhibition of the Cyclin-B-regulated signaling pathway involving p21 and Cdc2 expression. Additionally, expression of SPARC decreased STAT3 phosphorylation at Tyr-705; constitutively active STAT3 expression reversed SPARC induced G2/M arrest. Ad-DsRed-SP significantly inhibited the pre-established orthotopic tumor growth and tumor volume in nude-mice. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed decreased immunoreactivity for pSTAT3 and increased immunoreactivity for p21 compared to tumor section from mice treated with mock and Ad-DsRed. Taken together our studies further reveal that STAT3 plays a key role in SPARC induced G2/M arrest in medulloblastoma cells. These new findings provide a molecular basis for the mechanistic understanding of the effects of SPARC on medulloblastoma tumor cell proliferation.

Cerebral Microdialysis as a Tool for Assessing the Delivery of Chemotherapy in Brain Tumor Patients.

The development of curative treatment for glioblastoma has been extremely challenging. Chemotherapeutic agents that have seemed promising have failed in clinical trials. Drugs that can successfully target cancer cells within the brain must first traverse the brain interstitial fluid. Cerebral microdialysis (CMD) is an invasive technique in which interstitial fluid can be directly sampled. CMD has primarily been used clinically in the setting of head trauma and subarachnoid hemorrhage. Our goal was to review the techniques, principles, and new data pertaining to CMD to highlight its use in neuro-oncology. We conducted a literature search using the PubMed database and selected studies in which the investigators had used CMD in either animal brain tumor models or clinical trials. The references were reviewed for additional information. Studies of CMD have shown its importance as a neurosurgical technique. CMD allows for the collection of pharmacokinetic data on drug penetrance across the blood-brain barrier and metabolic data to characterize the response to chemotherapy. Although no complications have been reported, the current CMD technique (as with any procedure) has risks and limitations, which we have described in the present report. Animal CMD experiments have been used to exclude central nervous system drug candidates from progressing to clinical trials. At present, patients undergoing CMD have been monitored in the intensive care unit, owing to the requisite tethering to the apparatus. This can be expected to change soon because of advances in microminiaturization. CMD is an extremely valuable, yet underused, technique. Future CMD applications will have central importance in assessing drug delivery to tumor cells in vivo, allowing a pathway to successful therapy for malignant brain tumors.

MMP-2 alters VEGF expression via alphaVbeta3 integrin-mediated PI3K/AKT signaling in A549 lung cancer cells.

Vascular endothelial growth factor (VEGF) is one of the most important angiogenic growth factors for tumor angiogenesis. Here, we sought to explore whether RNA interference (RNAi) targeting matrix metalloproteinase-2 (MMP-2) could disrupt VEGF-mediated angiogenesis in lung cancer. MMP-2 siRNA inhibited lung cancer cell-induced tube formation of endothelial cells in vitro; addition of recombinant human-MMP-2 restored angiogenesis. MMP-2 transcriptional suppression decreased VEGF, phosphatidylinositol 3-kinase (PI3K) protein levels and AKT phosphorylation in lung cancer cells. In addition, MMP-2 suppression decreased hypoxia inducible factor-1alpha (HIF-1alpha), a transcription factor for VEGF, as determined by electrophoretic mobility shift assay (EMSA). We also show that MMP-2 suppression disrupted PI3K dependent VEGF expression; ectopic expression of myr-AKT restored VEGF inhibition. Further, MMP-2 suppression decreased the interaction of integrin-alphaVbeta3 and MMP-2 as confirmed by immunoprecipitation analyses. Studies with either function blocking integrin-alphaVbeta3 antibody or MMP-2 specific inhibitor (ARP-100) indicate that suppression of MMP-2 decreased integrin-alphaVbeta3-mediated induction of PI3K/AKT leading to decreased VEGF expression. Moreover, A549 xenograft tissue sections from mice that treated with MMP-2 siRNA showed reduced expression of VEGF and the angiogenic marker, factor-VIII. The inhibition of tumor angiogenesis in MMP-2 suppressed tumor sections was associated with decreased co-localization of integrin-alphaVbeta3 and MMP-2. In summary, these data provide new insights into the mechanisms underlying MMP-2-mediated VEGF expression in lung tumor angiogenesis.

Tumor Development and Angiogenesis in Adult Brain Tumor: Glioblastoma.

Angiogenesis is the growth of new capillaries from the preexisting blood vessels. Glioblastoma (GBM) tumors are highly vascularized tumors, and glioma growth depends on the formation of new blood vessels. Angiogenesis is a complex process involving proliferation, migration, and differentiation of vascular endothelial cells (ECs) under the stimulation of specific signals. It is controlled by the balance between its promoting and inhibiting factors. Various angiogenic factors and genes have been identified that stimulate glioma angiogenesis. Therefore, attention has been directed to anti-angiogenesis therapy in which glioma proliferation is inhibited by inhibiting the formation of new tumor vessels using angiogenesis inhibitory factors and drugs. Here, in this review, we highlight and summarize the various molecular mediators that regulate GBM angiogenesis with focus on recent clinical research on the potential of exploiting angiogenic pathways as a strategy in the treatment of GBM patients.

Rotating Magnetic Nanoparticle Clusters as Microdevices for Drug Delivery.

BACKGROUND: Magnetic nanoparticles (MNPs) hold promise for enhancing delivery of therapeutic agents, either through direct binding or by functioning as miniature propellers. Fluid-filled conduits and reservoirs within the body offer avenues for MNP-enhanced drug delivery. MNP clusters can be rotated and moved across surfaces at clinically relevant distances in response to a rotating magnet. Limited data are available regarding issues affecting MNP delivery by this mechanism, such as adhesion to a cellular wall. Research reported here was initiated to better understand the fundamental principles important for successful implementation of rotational magnetic drug targeting (rMDT). METHODS: Translational movements of four different iron oxide MNPs were tested, in response to rotation (3 Hz) of a neodymium-boron-iron permanent magnet. MNP clusters moved along biomimetic channels of a custom-made acrylic tray, by surface walking. The effects of different distances and cellular coatings on MNP velocity were analyzed using videography. Dyes (as drug surrogates) and the drug etoposide were transported by rotating MNPs along channels over a 10 cm distance. RESULTS: MNP translational velocities could be predicted from magnetic separation times. Changes in distance or orientation from the magnet produced alterations in MNP velocities. Mean velocities of the fastest MNPs over HeLa, U251, U87, and E297 cells were 0.24 ± 0.02, 0.26 ± 0.02, 0.28 ± 0.01, and 0.18 ± 0.03 cm/sec, respectively. U138 cells showed marked MNP adherence and an 87.1% velocity reduction at 5.5 cm along the channel. Dye delivery helped visualize the effects of MNPs as microdevices for drug delivery. Dye delivery by MNP clusters was 21.7 times faster than by diffusion. MNPs successfully accelerated etoposide delivery, with retention of chemotherapeutic effect. CONCLUSION: The in vitro system described here facilitates side-by-side comparisons of drug delivery by rotating MNP clusters, on a human scale. Such microdevices have the potential for augmenting drug delivery in a variety of clinical settings, as proposed.

Etoposide-Bound Magnetic Nanoparticles Designed for Remote Targeting of Cancer Cells Disseminated Within Cerebrospinal Fluid Pathways.

Magnetic nanoparticles (MNPs) have potential for enhancing drug delivery in selected cancer patients, including those which have cells that have disseminated within cerebrospinal fluid (CSF) pathways. Here, we present data related to the creation and in vitro use of new two-part MNPs consisting of magnetic gold-iron alloy cores which have streptavidin binding sites, and are coated with biotinylated etoposide. Etoposide was chosen due to its previous use in the CSF and ease of biotinylation. Etoposide magnetic nanoparticles ("Etop-MNPs") were characterized by several different methods, and moved at a distance by surface-walking of MNP clusters, which occurs in response to a rotating permanent magnet. Human cell lines including D283 (medulloblastoma), U138 (glioblastoma), and H2122 (lung adenocarcinoma) were treated with direct application of Etop-MNPs (and control particles), and after remote particle movement. Cell viability was determined by MTT assay and trypan blue exclusion. Results indicated that the biotinylated etoposide was successfully bound to the base MNPs, with the hybrid particle attaining a maximum velocity of 0.13 ± 0.018 cm/sec. Etop-MNPs killed cancer cells in a dose-dependent fashion, with 50 ± 6.8% cell killing of D283 cells (for example) with 24 h of treatment after remote targeting. U138 and H2122 cells were found to be even more susceptible to the killing effect of Etop-MNPs than D283 cells. These findings indicate that the novel Etop-MNPs have a cytotoxic effect, and can be moved relatively rapidly at physiologic distances, using a rotating magnet. While further testing is needed, intrathecal administration of Etop-MNPs holds promise for magnetically-enhanced eradication of cancer cells distributed within CSF pathways, particularly if given early in the course of the disease.

Inhibition of matrix metalloproteinase-2 enhances radiosensitivity by abrogating radiation-induced FoxM1-mediated G2/M arrest in A549 lung cancer cells.

Matrix metalloproteinase-2 (MMP-2), is known to degrade the collagen IV, plays a role in radiation-induced lung injury. We therefore investigated the antitumor effects of combining MMP-2 inhibition using an adenovirus expressing siRNA against MMP-2 (Ad-MMP-2-Si) with radiation therapy (IR) on A549 lung cancer cells in vitro and in vivo. IR increased MMP-2 mRNA, protein and activity in lung cancer cells. MMP-2 inhibition along with IR enhanced radiosensitivity as determined by clonogenic assay, flow cytometry and TUNEL assay. We show that MMP-2 inhibition prior to irradiation reduced p53 phosphorylation, with a corresponding reduction in the expression of the p53 downstream target gene p21(Cip1/Waf1). Irradiated tumor cells induced the FoxM1-mediated DNA repair gene, XRCC1 and Checkpoint kinases 2/1, which were abrogated with combined treatment of Ad-MMP-2-Si and IR. Further, the combination of Ad-MMP-2-Si with radiotherapy significantly increased antitumor efficacy in vivo compared to either agent alone. Indeed, histological analysis of tumor sections collected from the combination group revealed more apoptotic cells. These studies suggest that MMP-2 inhibition in combination with radiotherapy abrogates G2 cell cycle arrest leading to apoptosis and provide evidence of the antitumor efficacy of combining MMP-2 inhibition with irradiation as a new therapeutic strategy for the effective treatment of NSCLC patients.

Stat3-siRNA induces Fas-mediated apoptosis in vitro and in vivo in breast cancer.

Stat3, a member of the signal transducer and activator of transcription family, has the potential to mediate cell survival, growth and differentiation. Stat3 is constitutively activated in numerous cancers, including >50% of breast cancers. Previous studies demonstrated that constitutively activated Stat3 plays an important role in breast cancer development and progression by promoting cell proliferation and inhibiting apoptosis. The present study was designed to investigate the potential use of RNA interference (RNAi) to block Stat3 expression and activation, as well as the subsequent effect on human breast cancer cell growth. Our studies show that knockdown of STAT3 expression by siRNA reduced expression of Bcl-xL and survivin in MDA-MB-231 cells, and also led to Fas mediated intrinsic apoptotic pathway by activating caspases -8, -9, -3 and PARP1 cleavage. In nude mice, pRNAi-Stat3 significantly suppressed tumor growth compared with controls. It also suppressed Stat3 expression, and downregulated BcL-xL and upregulated Fas, Fas-L and cleaved caspase-3 expression within the tumor, which significantly induced apoptosis and led to tumor suppression. Thus, targeting Stat3 signaling using siRNA may serve as a novel therapeutic strategy for the treatment of breast cancers expressing constitutively activated Stat3.

Matrix metalloproteinase-9 inhibition down-regulates radiation-induced nuclear factor-kappa B activity leading to apoptosis in breast tumors.

PURPOSE: Novel strategies are needed to prevent the high mortality rates of several types of cancer. These high rates stem from tumor resistance to radiation therapy, which is thought to result from the induction of matrix metalloproteinases (MMP) and plasminogen activators. In the present study, we show that the modulation of MMP-9 expression, using adenoviral-mediated transfer of the antisense MMP-9 gene (MMP-9 adenoviral construct, Ad-MMP-9), affects breast cancer sensitivity to radiation. EXPERIMENTAL DESIGN: In the present study, we used antisense Ad-MMP-9 to down-regulate the expression of MMP-9 in MDA MB 231 breast cancer cell lines in vitro before irradiation and subsequently incubated cells in hypoxic condition. In vivo studies were done with orthotopic breast tumors, and radiosensitivity was evaluated both in vitro and in vivo. RESULTS: Ad-MMP-9 infection resulted in down-regulation of radiation-induced levels of hypoxia-inducible factor 1 alpha and MMP-9 under hypoxic conditions in MDA MB 231 breast cancer cells. In addition, Ad-MMP-9, in combination with radiation, decreased levels of the transcription factors nuclear factor-kappaB and activator protein 1, both of which contribute to the radioresistance of breast tumors. Finally, the triggering of the Fas-Fas ligand apoptotic cascade, which resulted in the cleavage of PARP-1 and caspase-10, caspase-3, and caspase-7, signifies the efficiency of combined treatment of Ad-MMP-9 and radiation. Treatment with Ad-MMP-9 plus radiation completely regressed tumor growth in orthotopic breast cancer model. CONCLUSIONS: In summary, integrating gene therapy (adenovirus-mediated inhibition of MMP-9) with radiotherapy could have a synergistic effect, thereby improving the survival of patients with breast cancer.

MMP-9 short interfering RNA induced senescence resulting in inhibition of medulloblastoma growth via p16(INK4a) and mitogen-activated protein kinase pathway.

The involvement of matrix metalloproteinases (MMP) has been suggested in cellular mechanisms leading to medulloblastoma, the most common malignant brain tumor in children. A significant association of the expression levels of MMP-9 with survival and M stage suggests that patients with medulloblastoma metastatic disease at diagnosis may benefit from the anti-MMP therapy. Here, we have evaluated the tumorigenicity of medulloblastoma cells after infection with an adenovirus containing a 21-bp short interfering RNA sequence of the human MMP-9 gene (Ad-MMP-9). Infection of Daoy medulloblastoma cells with Ad-MMP-9 reduced MMP-9 activity and protein levels compared with parental and Ad-SV controls. Ad-MMP-9 decreased the number of viable Daoy cells in a concentration-dependent manner. Fluorescence-activated cell sorting analysis indicated that Ad-MMP-9 infection caused a dose-dependent cell cycle arrest in the G(0)-G(1) phase. Ad-MMP-9-induced cell cycle arrest seems to be mediated by the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway and the cell cycle inhibitor p16(INK4a) and is phenotypically indistinguishable from senescence. Ad-MMP-9 treatment inhibited medulloblastoma tumor growth in an intracranial model and was mediated by up-regulation of p16 expression. These studies validate the usefulness of targeting MMP-9 and provide a novel perspective in the treatment of medulloblastoma.

Elucidating the microRNA-203 specific biological processes in glioblastoma cells from comprehensive RNA-sequencing transcriptome profiling.

Glioblastoma (GBM) is the most common primary malignant intracranial adult brain tumor. Allelic deletion on chromosome 14q plays an essential role in GBM pathogenesis, and this chromosome 14q site was thought to harbor multiple tumor suppressor genes associated with GBM, a region that also encodes microRNA-203 (miR-203). This study was conducted to identify whole transcriptome profile changes associated with miR-203 expression by high-throughput RNA sequencing. Enrichment analyses for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that miR-203 expression had a strong, negative effect on a number of fundamental and interconnected biological processes involved in cell growth and proliferation. The biological processes mostly influenced were p53 signaling pathway, FoxO signaling pathway, DNA replication, cell cycle, MAPK signaling pathway, and apoptosis. In total, 847 upregulated and 345 downregulated differentially expressed genes were identified in control versus miR-203 expressing glioma cells. After GO enrichment, the downregulated differentially expressed genes such as BCL2, SPARC were found to be mainly enriched in cell cycle regulation and apoptosis processes, whereas the upregulated differentially expressed genes such as CCND1, E2F1 were involved in the DNA replication and cell cycle regulation. We also performed miR-203 target analysis and found BCL2, AKT, SPARC, ROBO1, c-JUN, PDGFA, and CREB were predicted target of miR-203 and miR-203 expression suppressed the protein and mRNA levels of these target genes by western blotting and qRT-PCR analysis. Moreover, co-transfection experiments using a luciferase-based reporter assay demonstrated that miR-203 directly regulated BCL-2 expression and BCL-2 overexpression suppressed miR-203 mediated glioma cell apoptosis. These results indicate that overexpression of miR-203 coordinately regulates several oncogenic pathways in GBM.

Downregulation of matrix metalloproteinase-2 (MMP-2) utilizing adenovirus-mediated transfer of small interfering RNA (siRNA) in a novel spinal metastatic melanoma model.

Matrix metalloproteinases (MMPs) comprise a class of secreted zinc-dependent endopeptidases implicated in the metastatic potential of tumor cells due to their ability to degrade the extracellular matrix (ECM) and basement membrane. Matrix metalloproteinase-2 (MMP-2) has been detected in high levels and correlates with invasiveness in human melanoma. We have studied the effect of adenovirus-mediated transfer of small interfering RNA (siRNA) against MMP-2 in the human melanoma cell line A2058. The delivery of these double-stranded RNA molecules represents an efficient technology in silencing disease-causing genes with known sequences at the post-transcriptional level. siRNA against MMP-2 mRNA (Ad-MMP-2) was found to decrease MMP-2 protein expression and activity in melanoma cells as demonstrated by western blotting and gelatin zymography. Furthermore, infection of cells with Ad-MMP-2 inhibited cellular migration and invasion as indicated by spheroid and matrigel assays. We also observed dose-dependent suppression of vascular network formation in an angiogenesis assay. Finally, we developed a nude mouse spinal metastatic model to investigate the local effects of tumor metastasis. Intravenous tail vein injection with Ad-MMP-2 on days 5, 9 and 11 after tumor implantation resulted in complete retention of neurological function as compared to control and scrambled vector (Ad-SV)-treated groups that showed complete paraplegia by day 14+/-2 days. Hematoxylin and eosin staining revealed decreased tumor size in the Ad-MMP-2-treated animals. This novel experimental model revealed that adenoviral-mediated transfer of RNA interference against MMP-2 results in the retention of neurological function and significantly inhibited tumor growth.

Intraperitoneal injection of a hairpin RNA-expressing plasmid targeting urokinase-type plasminogen activator (uPA) receptor and uPA retards angiogenesis and inhibits intracranial tumor growth in nude mice.

PURPOSE: The purpose of this study was to evaluate the therapeutic potential of using plasmid-expressed RNA interference (RNAi) targeting urokinase-type plasminogen activator (uPA) receptor (uPAR) and uPA to treat human glioma. EXPERIMENTAL DESIGN: In the present study, we have used plasmid-based RNAi to simultaneously down-regulate the expression of uPAR and uPA in SNB19 glioma cell lines and epidermal growth factor receptor (EGFR)--overexpressing 4910 human glioma xenografts in vitro and in vivo, and evaluate the i.p. route for RNAi-expressing plasmid administered to target intracranial glioma. RESULTS: Plasmid-mediated RNAi targeting uPAR and uPA did not induce OAS1 expression as seen from reverse transcription-PCR analysis. In 4910 EGFR-overexpressing cells, down-regulation of uPAR and uPA induced the down-regulation of EGFR and vascular endothelial growth factor and inhibited angiogenesis in both in vitro and in vivo angiogenic assays. In addition, invasion and migration were inhibited as indicated by in vitro spheroid cell migration, Matrigel invasion, and spheroid invasion assays. We did not observe OAS1 expression in mice with preestablished intracranial tumors, which were given i.p. injections of plasmid-expressing small interfering RNA--targeting uPAR and uPA. Furthermore, the small interfering RNA plasmid targeting uPAR and uPA caused regression of preestablished intracranial tumors when compared with the control mice. CONCLUSION: In conclusion, the plasmid-expressed RNAi targeting uPAR and uPA via the i.p. route has potential clinical applications for the treatment of glioma.

RNAi-mediated downregulation of urokinase plasminogen activator receptor and matrix metalloprotease-9 in human breast cancer cells results in decreased tumor invasion, angiogenesis and growth.

The serine protease urokinase-type plasminogen activator (uPA) plays a significant role in tumor cell invasion and metastasis when bound to its specific receptor, uPAR (also known as CD87). In addition to the uPA-uPAR system, matrix metalloproteinases (MMPs) are involved in tumor cell invasion and metastasis. In this study, we achieved specific inhibition of uPAR and MMP-9 using RNAi technology. We introduced small interfering RNA to downregulate the expression of uPAR and MMP-9 (pUM) in breast cancer cell lines (MDA MB 231 and ZR 75 1). In vitro angiogenesis studies indicated a decrease in the angiogenic potential of the treated cells; in particular, a remarkable decrease was observed in the cells treated with bicistronic construct (pUM) in comparision to the controls. Additionally, bicistronic construct inhibited the formation of capillary-like structures in in vivo models of angiogenesis. Similarly, the invasive potential and migration decreased dramatically when treated with the bicistronic construct as shown by matrigel invasion and migration assays. These results suggest a synergistic effect from the simultaneous downregulation of uPAR and MMP-9. We also assessed the levels of phosphorylated forms of MAPK, ERK and AKT signaling pathway molecules and found reduction in the levels of these molecules in cells treated with the bicistronic construct as compared to the control cells. Furthermore, targeting both uPAR and MMP-9 totally regressed orthotopic breast tumors in nude mice. In conclusion, our results provide evidence that the simultaneous downregulation of uPAR and MMP-9 using RNAi technology may provide an effective tool for breast cancer therapy.